Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma.

Interactions between tumour cells and surrounding extracellular matrix (ECM) influence the growth of tumour cells and their ability to metastasise. It is thus interesting to compare ECM composition in tumours and healthy tissues. Using the recently described MeLiM miniature pig model of heritable cutaneous malignant melanoma, we studied the expression of two ECM glycoproteins, the tenascin-C (TN-C) and tenascin-X (TN-X), in normal skin and melanoma. Using semiquantitative RT-PCR, we observed a 3.6-fold mean increase of TN-C RNAs in melanoma compared to normal skin. Both stromal and tumour cells synthesise TN-C. On the contrary, TN-X RNAs decreased 30-fold on average in melanoma. This opposite regulation of TN-C and TN-X RNAs was confirmed at the protein level by indirect immunofluorescence. Whereas pig normal skin displayed a discrete TN-C signal at the dermo-epidermal junction, around blood vessels and hair bulbs, the swine tumour showed enhanced expression of TN-C in these areas and around stromal and tumour cells. In contrast, normal skin showed a strong TN-X staining at the dermo-epidermal junction and in the dermis, whereas this signal almost completely disappeared in the tumour. The results presented here describe a dramatic alteration of the ECM composition in swine malignant melanoma which might have a large influence on tumourigenesis or invasion and metastasis of melanoma cells.

Histopathological and cellular studies of a case of cutaneous radiation syndrome after accidental chronic exposure to a cesium source.

This study was designed for the histopathological, cellular and biochemical characterization of a skin lesion removed surgically from a young male several months after accidental exposure to cesium-137, with an emphasis on expression of transforming growth factor beta1 (TGFB1) and tumor necrosis factor alpha (TNFA) and the occurrence of apoptosis. Under a hypertrophic epidermis, a highly inhomogeneous inflammatory dermis was observed, together with fibroblastic proliferation in necrotic areas. Immunostaining revealed overexpression of TGFB1 and TNFA inside the keratinocytes of the hypertrophic epidermis as well as in the cytoplasm of the fibroblasts and connective tissue of the mixed fibrotic and necrotic dermis. Inside this dermis, the TUNEL assay revealed areas containing numerous apoptotic fibroblasts next to areas of normal viable cells. Overexpression of TGFB1 was found in the conditioned medium and cellular fractions of both hypertrophic keratinocytes and fibrotic fibroblasts. This overexpression lasted for at least three passages in tissue culture. The present observations were consistent with the central role of TGFB1 in the determination of chronic radiation-induced damage to the skin and a significant involvement of TNFA. In addition, programmed cell death appeared to take place during the remodeling of the mixed fibrotic and necrotic tissue.

Striking regression of subcutaneous fibrosis induced by high doses of gamma rays using a combination of pentoxifylline and alpha-tocopherol: an experimental study.

PURPOSE: To establish a successful treatment of subcutaneous fibrosis developing after high doses of gamma rays, suitable for use in clinical practice. METHODS AND MATERIALS: We used an animal model of acute localized gamma irradiation simulating accidental overexposure in humans. Three groups of 5 Large White pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of necrosis developed within a few weeks which had healed after 26 weeks to leave a block of subcutaneous fibrosis involving skin and skeletal muscle. One experimental group of 5 pigs was dosed orally for 26 weeks starting 26 weeks after irradiation with 1600 mg/120 kg body weight of pentoxifylline (PTX) included in the reconstituted food during its fabrication, and another group of 5 was dosed orally for the same period with a daily dose of 1600 mg/120 kg body weight of PTX combined with 2000 IU/120 kg body weight of alpha-tocopherol. Five irradiated control pigs were given normal food only. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Depth of scar tissue was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 26 weeks after treatment started. The density, length, width, and depth of the block of fibrotic scar tissue, and the areas and volume of its projected cutaneous surface, were compared before treatment, 6 and 13 weeks thereafter, and at 26 weeks. RESULTS: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. No modifications were observed in the block of fibrotic scar tissue of pigs dosed with PTX alone. However, significant softening and shrinking of this block were noted in the pigs dosed with PTX + alpha-tocopherol 13 weeks after treatment started and at autopsy, when mean regression was approximately 30% for length, approximately 50% for width and depth, and approximately 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar tissue. The 50% decrease in the linear dimensions of the scar tissue, were comparable to the results obtained in our previous clinical studies, and were highly significant compared to the clinical and autopsy results for the controls. Histologic examination of the residual scar tissue revealed tissue which was more homogenous and less cellular and inflammatory than in control and PTX-dosed pigs. The tissular and cellular immunolocalization of tumor necrosis factor alpha (TNFalpha) was similar in the residual fibrotic tissues of all three groups of pigs, whereas the immunostaining of transforming growth factor beta-1(TGFbeta-1) diminished much more in the residual fibrotic scar tissue of the PTX + alpha-tocopherol-dosed pigs than in the two other groups. CONCLUSIONS: The present results showed a striking regression of the subcutaneous fibrotic scar tissue that develops as a consequence of high doses of gamma rays.

Unlike tenascin-X, tenascin-C is highly up-regulated in pig cutaneous and underlying muscle tissue developing fibrosis after necrosis induced by very high-dose gamma radiation.

Fibrosis is characterized by proliferation of fibroblasts and deposition of extracellular matrix (ECM). As alterations in the composition of ECM may account for its chronic extension, we studied the expression of the tenascin-C (TN-C) and tenascin-X (TN-X) ECM glycoproteins in our pig model of the effects of accidental exposures to radiation, in which cutaneous and muscle fibrosis developed after the induction of necrosis after a high single dose (160 Gy at the skin surface) of gamma rays. We found that, in the healed fibrotic dermis and underlying muscle fibrosis, the amount of TN-C mRNA was increased up to 18- and 39-fold, respectively, compared to normal dermis, whereas the level of TN-X mRNA remained almost unchanged. In analyses by Western blotting, the two main TN-C isoforms of 235-240 and 190-200 kDa increased up to 45- and 105-fold in fibrotic tissues, respectively. The large isoform was expressed more strongly than the smaller, although in healed fibrotic scar tissues their ratio was lower in protein than in RNA. Compared to unirradiated skin, an immunohistological study revealed stronger TN-C staining at the dermo-epidermal junction and in areas of remodeling in healed skin. An intense extracellular staining was observed around myofibroblasts in muscle fibrosis. Therefore, the gene encoding TN-C is highly up-regulated in fibrotic tissues, and mechanisms regulating the levels of TN-C variants occur at both the RNA and protein levels. Each isoform might play a distinct role in the chronic activation of fibrosis by differentially regulating mechanisms like cell adhesion, migration or proliferation.

Dosimetry of 60Co and 192Ir gamma-irradiated agarose gels by proton relaxation time measurement and NMR imaging, in a 0-100 Gy dose range.

Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma rays can induce immediate effects characterized by erythema, desquamation, and necrosis. Correlations between the evolution of the lesions and dosimetry studies have to be established by biophysical methods. NMR studies of the effects of an irradiated Fricke solution might be a means of controlling the delivered irradiation doses. After exposition to ionizing radiations, ferrous ions are transformed into ferric ions. Both are paramagnetic ions, and proton spin-lattice relaxation is accelerated depending on the oxidation reaction. In this study, solution of ammonium ferrous sulfate in an acid environment was incorporated into a gelling substance made with agarose, so that T1 weighted image contrast could be used to detect ferric ion formation. Experiments with 192Ir and 60Co gamma rays with doses in the 0 to 100 Gy range were conducted with Fe2+ concentrations of 0.5, 1, 1.5, and 2 mM in a gelling substance containing 4% agarose. A relationship was established between the amount of Fe3+ created and the spin-lattice proton relaxation rate, which led to a straightforward dose-effect relation. The use of such high doses allowed us to reproduce realistic conditions of accidental overexposure. A linear relationship was obtained between the doses absorbed and the NMR parameters measured (T1 and relative image intensity).

Successful treatment of radiation-induced fibrosis using Cu/Zn-SOD and Mn-SOD: an experimental study.

PURPOSE: To establish how far liposomal copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD), respectively, reduce radiation-induced fibrosis (RIF), using a well-characterized pig model of RIF permitting the design of a controlled laboratory experiment. METHODS AND MATERIALS: In this model of acute localized gamma irradiation simulating accidental overexposure in humans, three groups of five large white pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of subcutaneous fibrosis involving skin and skeletal muscle developed 6 months after irradiation. One experimental group of five pigs was then injected i.m. with 10 mg/10 kg b.wt. of Cu/Zn-SOD, twice a week for 3 weeks, and another experimental group of five was injected with 10 mg/10 kg b.wt. of Mn-SOD, three times a week for 3 weeks. Five irradiated control pigs were injected with physiological serum. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Block depth was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 12-14 weeks after completing SOD injections. The density, length, width, and depth of the fibrotic block, and the areas and volume of its projected cutaneous surface were compared before treatment, 1, 3, and 6 weeks thereafter, and at autopsy, 12-14 weeks after treatment ended. RESULTS: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. Whether they were given Cu/Zn- or Mn-SOD, significant and roughly equivalent softening and shrinking of the fibrotic block were noted in all treated animals between the first week after treatment ended and autopsy, when mean regression was 45% for length and width, 30% for depth, and 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissue in experimental animals and the 50% decrease in the linear dimensions of the scar were comparable to the results obtained in previous clinical studies and highly significant compared to the clinical and autopsy results for the control animals. CONCLUSIONS: Our results are striking and comparable to the results obtained in our previous clinical study after liposomal Cu/Zn-SOD treatment. To our knowledge, this is the first time that two agents have been shown to reverse the radiation-induced fibrotic process in experimental animals and to permit the regeneration of normal tissue in a zone of well-established postirradiation fibrosis.

[Radiation-induced cutaneo-muscular fibrosis (III): major therapeutic efficacy of liposomal Cu/Zn superoxide dismutase]. / La fibrose cutanéo-musculaire radio-induite (III): efficacité thérapeutique majeure de la superoxyde dismutase Cu/Zn liposomiale.

Sub-cutaneous and muscular fibrosis are common and irreversible late effect of radiation on normal tissues. An experiment was designed to test the effectiveness of superoxide dismutase in reducing late radiation injury. This study was performed in an experimental porcine model of acute localized gamma irradiation simulating human accidental overexposure: 12 Large White pigs were irradiated on the thigh with a collimated gamma 192Ir source, so that the dose was 160 Gy/skin (100%) and 40 Gy/2 cm depth (25%). In this model, fibrosis appears in 4 to 5 months. The heterogeneous sclerotic tissue is composed of stable fibrotic areas poorly cellularized and active areas with a high density of myofibroblasts and inflammatory perifibrotic part. Lipsod administration modalities were six intramuscular injections during 3 weeks (twice weekly) either 10 mg/inj (five pigs) or of 100 mg/inj (five pigs). A methodic evaluation by two examiners consisted of measurements being taken before and after treatment: sum of the two largest perpendicular measurable dimensions, cutaneous projected surface of palpated fibrotic block, ultrasound fibrosis deepness and extrapolated volume. We conclude that Lipsod is the first drug ever described that reduces radiation-induced fibrosis. Its efficacy in this model was highly significant, with a regression higher than 40% in size and 70% in surface and volume, 12 weeks after the end of treatment. This response was rapid, reproducible without dose-effect or toxicity in the limits studied. This work confirms previously published results in humans.

Muscular fibrosis induced after pig skin irradiation with single doses of 192Ir gamma-rays.

Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma-rays can induce delayed effects characterized by fibrosis which invades the irradiated tissues. In this study the depth of penetration of muscle fibrosis was measured in the pig 30 weeks after irradiation of the skin surface with single doses of 192Ir gamma-rays of 16-256 Gy. Irradiation was directed either to the outer side of the thigh or to the back, close to the mid-dorsal line. Fibrosis only developed in irradiated muscle after doses that induced moist desquamation of the skin in the acute phase of the reaction, i.e. after skin surface doses of 48-64 Gy. In skeletal muscles, the limit of fibrotic expansion was reached at a depth dose of 14 +/- 4 Gy (+/- SD) for skin surface doses exceeding 48 Gy.

Caesium and iodine metabolism in lactating cows under chronic administration.

1) Two Jersey cows were fed during 90 days with contaminated hay harvested in the South-East of France and containing about 5500 Bq/kg of dry matter (134 Cs + 137 Cs). A plateau was observed in milk 15 days and in meat 50-60 days after the beginning of the contamination. The transfer coefficients were at that time 1.1% for the milk and 2-2.7% for the meat. A calf was fed from the 8th day after birth until the 80th day solely with the contaminated milk from the two cows. At that time and weighing 130 kg it retained more than 40% of the ingested caesium. From the 44th till the 80th day the daily intake of milk was constant: in these conditions the transfer coefficient to meat was 16%. 2) Transfer coefficients to milk and thyroid were determinated in three Friesian cows contaminated daily with Na131I in pellets, one month before and two months after calving; one more lactating cow was contaminated for one month, 5 months after calving. The parturition provokes a fall of about 40% in the thyroid burden of the cows followed by a return near the initial equilibrium level (2-3 times the daily uptake in summer and 1 time in winter). At birth the ratio between the specific radioactivity of the calf thyroid and the cow thyroid was about 3. Transfer coefficients in milk at the equilibrium (one month after calving) were 0.35-0.50% in summer and 0.75-1.25 in winter.